Design, Synthesis, and Biological Evaluation of Small, High-Affinity

Article pubs.acs.org/jmc

Design, Synthesis, and Biological Evaluation of Small, High-Affinity Siglec‑7 Ligands: Toward Novel Inhibitors of Cancer Immune Evasion Horst Prescher,§,‡ Martin Frank,¶,‡,† Stephan Gütgemann,∥,‡ Elena Kuhfeldt,§ Astrid Schweizer,⊥ Lars Nitschke,⊥ Carsten Watzl,∥,# and Reinhard Brossmer*,§,∇ §

G3-BioTec, 69207 Sandhausen, Germany Institute of Immunology and ∇Biochemistry Center, University of Heidelberg, 69120 Heidelberg, Germany ¶ Molecular Structure Analysis Core Facility-W160, German Cancer Research Center, 69120 Heidelberg, Germany ⊥ Division of Genetics, Department of Biology, University of Erlangen, 91058 Erlangen, Germany ∥

S Supporting Information *

ABSTRACT: Natural killer cells are able to directly lyse tumor cells, thereby participating in the immune surveillance against cancer. Unfortunately, many cancer cells use immune evasion strategies to avoid their eradication by the immune system. A prominent escape strategy of malignant cells is to camouflage themselves with Siglec-7 ligands, thereby recruiting the inhibitory receptor Siglec-7 expressed on the NK cell surface which subsequently inhibits NK-cell-mediated lysis. Here we describe the synthesis and evaluation of the first, high-affinity low molecular weight Siglec-7 ligands to interfere with cancer cell immune evasion. The compounds are Sialic acid derivatives and bind with low micromolar Kd values to Siglec-7. They display up to a 5000-fold enhanced affinity over the unmodified sialic acid scaffold αMe Neu5Ac, the smallest known natural Siglec-7 ligand. Our results provide a novel immuno-oncology strategy employing natural immunity in the fight against cancers, in particular blocking Siglec-7 with low molecular weight compounds.

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INTRODUCTION

ligands have been shown to have sufficient affinity to allow investigation of their effect on Siglec-7 functions.22,30−39 Nature compensates the low affinity of many lectins, including Siglec-7, with increased avidity obtained by polyvalent display of their ligands.40,41 No generally applicable approach to mimic the complex spatial and flexible display of natural, glycocalixassociated low-affine but polyvalent ligands has been described. A few reports elegantly solved the challenge of mimicking natural, polyvalent display of Siglec-7 ligands to investigate specific functions of the protein.15,16,36 Results from these studies together with additional medical, biological, and biochemical data provide strong evidence that Siglec-7 plays a role in cancer immune evasion.5,13,29−31,42−44 We aimed to develop soluble, monovalent high-affinity ligands to explore their effect on Siglec-7, especially since the effect of monovalent, low molecular weight compounds might be completely different compared to polyvalent ligands or antibodies. Here, we describe the design, synthesis, and biological evaluation of the first monovalent, low molecular weight Siglec7 ligands with sufficient affinity to investigate their impact on

Siglec-7 (p75/AIRM) belongs to the Siglec (sialic acid recognizing immunoglobulin-like lectins) family and recognizes specifically sialic acid-containing carbohydrates.1−5 The important roles of Siglecs in immunity have been recognized for many years.3,4,6−9 Recent research also showed disease implications and the potential to develop therapeutics targeting the lectin function of these proteins.10−16 In addition, an approach broadly targeting Siglecs and other Sialic acid binding lectins with a Sialic acid mimetic peptide has shown promising data that these receptors function as checkpoint inhibitors and could be therapeutically used to fight cancers.17 Natural killer cells (NK cells) express Siglec-7 at high levels, although the protein is found on other immune cells as well.2,5,14,18−25 Importantly, the effector functions of NK cells are regulated by the interplay of activating and inhibitory surface receptors.26,27 Siglec-7 acts as an inhibitory surface receptor via its intracellular immunoreceptor tyrosine-based inhibition motif (ITIM) and ITIM-like domains, providing the basis for the hypothesis that blocking the inhibition might result in enhanced cancer lysis activity.5,13,16,18,28−30 A range of different ligands, including natural ones like gangliosides, oligosaccharides, and modified sialic acids have been identified, but so far, no soluble, monovalent Siglec-7 © XXXX American Chemical Society

Received: July 27, 2016

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DOI: 10.1021/acs.jmedchem.6b01111 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Siglec-7 function. In addition, we show for the first time that low molecular weight compounds can be used to block Siglec7-mediated cancer immune evasion.

for the affinity improvement to mCD22.45,46 Compounds 9 and 10 containing 2-naphthylacetyl and phenylacetyl residues displayed affinities up to 10-fold higher than 1. They showed higher affinities than known 2, but did not improve over recently reported 3.34,39 In contrast, 8, substituted with 1naphtylacetyl, and 11, 12, and 13 with more rigid or aliphatic residues did not enhance affinity (Table 1). We were interested to explore the structure−activity relationship with computational methods to get some clues on how to modify the compounds better binding. To date, six different crystal structures of Siglec-7 have been reported, providing a good basis for docking experiments and MD simulations.34,49−52 An overlay of the available crystal structures revealed significant differences in the side-chain orientation of key amino acids in the vicinity of the Neu5Ac binding site, e.g., Lys135, Trp86, Arg 124, and Trp124, as well as major loop flexibility (Figure S1). The earlier identified hydrophobic gate in extension of the ligand’s glycerol side chain comprised of Lys-135 and Tyr-136 shows substantial flexibility of amino acid side-chains, but a quite rigid backbone (Figure S1).34,53 In addition, we found high flexibility in the extension of the glycosidic bond, including previously reported major conformational changes.50 This flexibility together with transient potential binding cavities (Figure 1) makes the interpretation and prediction of

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RESULTS AND DISCUSSION Chemistry and Ligand Design. It has been shown that aromatic substitutions at position 9 of sialic acids can substantially increase affinity to different Siglecs.34,45−48 Therefore, we synthesized a small array of novel and known α-Me Neu5Ac analogs and screened them for their affinity to Siglec-7 in cell-based assay39,45,46 (Table 1 and Scheme S1). Table 1. The rIPs of Known and New 9-N-Substituted Derivatives of α-O-Me Neu5Ac

Figure 1. Flexible side chains and cavities in proximity to the ligand binding side.

structure−activity relationships difficult and susceptible to misinterpretations. Nevertheless, we performed docking experiments with the aromatic substituted ligands 7, 9, and 10. Two representative PDB entries (2HRL and 2G5R) were selected as receptor models (Figure 1) to address receptor flexibility. Missing parts in 2HRL (residues 18−25 and 52−58) were taken from pdb entry 1O7V and the resulting hybrid receptor model was termed 2HRL*. Docking poses were generally in agreement with the expected binding mode of sialic acid (Figure 2a), defined mainly by the interactions of the ligand’s glycerol chain with Asn-133 and Trp-132 and a salt bridge between the ligand’s carboxylate and Arg-124, as determined by crystal structures.34,49,50 To our surprise, some poses with PDB entry 2HRL showed a completely flipped ligand with the biphenyl substituent located in the previously identified Tyr-64 cavity (Figure 2b).50 We designed virtual ligand 14 (Figure 3a) based on the above finding. The aromatic biphenyl fragment of 14 is connected with an aliphatic pentyl linker to the glycosidic oxygen at position 2 of scaffold 1 to allow a “natural” binding

Soluble, recombinant Siglec-7-Fc protein binds via its lectin domain to red blood cells (RBCs) surface-bound sialic acids,34 which we detected by flow cytometry.39 Soluble ligands bind competitively to Siglec-7 and inhibit binding to the RBCs, resulting in a decreased signal. The assay employs freshly isolated RBCs and therefore displays inter-test differences, which leads to high standard deviations of IC50 values.39 We used 1 as reference compound and calculated intra-test relative inhibitory potency (rIP) values and obtained reproducible values, as reported earlier.39 The ligands of MAG (Siglec-4) and hCD22 (Siglec-2) 5 and 6, substituted with benzoyl and 4biphenylcarbonyl, respectively, did not show improved binding to Siglec-7.45,46 Compound 7, substituted with 4-biphenylacetyl, resulted in a rIP of 4.5, much less than observed previously B



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Docking experiments confirmed the desired binding mode (Figure 3b). We decided to introduce a triazole into the linker to simplify synthetic efforts and reduce lipophilicity. Therefore, the 3chloropropyl glycoside 15 was crystallized to obtain the pure αanomer, which was transformed into the 3-azidopropyl glycoside (Scheme S2).54 Zemplén deacetylation and Huisgen triazole formation gave 16, which was converted into 17 upon saponification (Scheme 1). Scheme 1. Synthesis of 2-α-Biphenyltriazol 17a

Figure 2. (a) Exemplary docking result of PDB entry 2G5R with ligand 7. (b) Exemplary docking result of PDB entry 2HRL* with flipped ligand 7. a Reagents and conditions: (a) 15, DMF, NaN3, 80 °C, 80%. (b) 4Ethinyl-1,1-biphenyl, tert-butanol, DMF, H2O, sodium ascorbate, CuSO4, 99%. (c) MeOH, NaOMe, 90%. (d) EtOH, H2O, NaOH, 79%.

The affinity of 17 was found to be 152-fold higher compared to 1 (Table 2). Intriguingly, the discovery of 17 was driven by Table 2. The rIPs of α-O-Me Neu5Ac and 2-α-O-(Biphenyltriazolyl-propyl) Neu5Ac

careful interpretation of unexpected binding modes derived from docking experiments and subsequent shuffling of the aromatic substitution from position 9 to position 2. New docking experiments with 17 showed the expected binding mode (Figure S2), although alternative binding modes were found as well. We used 17 as the new reference compound for further design and affinity testing. The 9-hydroxyl group of sialic acid acts as a hydrogen-bond donor in known Siglec-7 ligands and removal of the capacity to

Figure 3. (a) Virtual ligand 14. (b) Exemplary docking result of PDB entry 2HRL* with ligand 14.

mode of the carbohydrate fragment of 1 and simultaneous stacking of the biphenyl fragment into the TYR-64 cavity. C



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Scheme 2. Exchange of the 9-Hydroxyl Groupa

Reagents and conditions: (a) (i) toluene, 0 °C, triphenylphosphine, diisopropyl azodicarboxylate; (ii) 16, DMF, 0 °C, tetrabutylammonium iodide, formic acid, 69%. (b) (i) EtOH, H2O, NaHCO3, Pd/C, H2; (ii) EtOH, H2O, NaOH, 72%. (c) (i) toluene, 0 °C, triphenylphosphine, diisopropyl azodicarboxylate; (ii) 16, DMF, 0 °C, thioacetic acid, 59%. (d) (i) MeOH, NaOMe; (ii) H2O, 24%. (e) 16, DMF, LiN3, CBr4, triphenylphosphine, 79%. (f) (i) MeOH, H2O, triphenylphosphine; (ii) HAc, 91%. (g) 20, EtOH, H2O, NaOH, 90% a

donate a hydrogen results in reduced binding.34,50,55 Therefore, we tested if the hydroxyl group at position 9 of 17 is also important for affinity to confirm the correctness of our predicted binding model. First, we synthesized the 9-deoxy analog 18. Selective replacement of the 9-hydroxyl by iodo group was achieved analogous to a previously published Mitsunobu-type procedure (Scheme 2).56 Subsequent removal of the iodine by hydrogenation with Pd/C and saponification produced the desired compound 18 (Scheme 2). The affinity decreased to a rIP of 0.15, indicating the importance of the hydrogen bond at position 9 (Table 3). Second, we replaced

procedures58 resulted in the acetate salt 20 (Scheme 2). Subsequent saponification produced the desired amine 21 (Scheme 2). The replacement of the hydroxyl by the amino group resulted in reduced binding (Table 3), potentially caused by protonation and repulsion of ε-NH2-Lys-135. We acetylated amine 20 and obtained 22 after subsequent saponification (Scheme 3). The acetylation of the amino group indeed resulted in a regain of binding, likely due to improved hydrogen-bond-donor properties and loss of the positive charge (Table 3). Furthermore, we synthesized the alkylated derivatives 23 and 24 via reductive alkylation with sodium cyan borohydride as well as squaric acid modified derivative 25 (Scheme 3). In contrast to the acetylation, no regain in affinity was observed (Table 3). Overall, the results show that the 9hydroxyl group plays an important role as a hydrogen-bond donor and confirm the binding mode predicted by our docking experiments. The confirmed binding model opened the way to explore how to modify 17 to enhance binding further. Previous reports have described oxamate and phenyloxamate substitutions at position 9 to enhance affinity to Siglec-7.34,39 Therefore, we expected augmented affinity upon introduction of these substituents at position 9 of 17. A first attempt to synthesize the novel oxamate analog 26 by activation of oxamic acid with HATU, coupling to amine 20, and subsequent saponification yielded the oxalic acid derivative 27. Coupling of the already saponified amine 21 with oxamic acid nitrophenylester produced the desired 26 (Scheme 3). Indeed, we found increased affinities for both compounds within the expected range (Table 4). The reason is most probably an additional hydrogen bond of the terminal oxamide carbonyl and the backbone nitrogen of Lys-135 as previously shown for oxamate 2.34 The results further consolidate the predicted binding mode. The phenyloxamate analog 29 was synthesized in analogy to a published procedure39 by exchanging the acetate salt 20 with the corresponding hydrochloride salt 28 followed by HATU-based coupling and subsequent saponification (Scheme 3). The affinity increased 6.8-fold (Table 4), almost 10 times less than the factor found for the corresponding α-Me Neu5Ac analog 3.39 In a recent report, we explained that the enhanced binding of 3 is composed of a fine balance of positive and negative contributors39 and that the 2-α-biphenyltriazol most probably causes an induced fit, which also changes the interactions at position 9.

Table 3. The rIPs Obtained by Derivatives with a Replaced Hydroxyl Group

the hydroxyl with a thiol group by introducing an acetylthio group and subsequent saponification under oxygen-free conditions (Scheme 2).57 The affinity assay showed a rIP of 0.52, indicating a bioisosteric replacement of the hydrogen donor capacity with minor effects on affinity (Table 3). Third, we replaced the hydroxyl with an amine. Introduction and reduction of an azide analogous to recently reported D



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Scheme 3. Substitutions of the 9-Amino Groupa

a

Reagents and conditions: (a) 20, DMF, DIPEA, HATU, AcOH. (b) EtOH, H2O, NaOH, 80% (a plus b). (c) (i) 20, MeOH, aldehyde, 1 h; (ii) NaCNBH4, 17 h, 72% and 27%, respectively (c plus b). (d) 20, DMF, diethyl squaric acid, triethylamine, 98% (d plus b). (e) 28, DMF, triethylamine, oxamic acid 4-nitrophenyl ester, 94% (e plus b). (f) (i) DMF, DIPEA, HATU, oxamic acid; (ii) 20, 39% (f plus b). (g) 28, DMF, phenyloxamic acid, HATU, DIPEA, 55% (g plus b). (h) (i) DMF, DIPEA, HATU, phenylacetic acid; (ii) 20, 52% (i plus b).

combined with direct saponification (Scheme 4). To our delight, the affinity increased about 33-fold over 17 (Table 5). To test the importance of the negative charge, we aimed to synthesize the uncharged methylsulfonyl containing bioisostere 32 (Table 6). The reactive methanesulfonyl chloride did react exclusively with the acetate ions of 20 and subsequently acetylated the amine of 20, resulting in N-acetylated 22 upon saponification. Therefore, we transformed the acetate salt 20 into hydrochloride salt 28 and obtained the desired 32 upon reaction of 28 with methanesulfonyl chloride and subsequent saponification (Scheme 4). Surprisingly, and in conflict with our hypothesis, the rIP value remained unchanged and high, and the affinity gain must have another basis than the predicted ionic interaction (Table 6). Docking experiments showed a binding mode analogous to the previously found ones (Figure 4) without interaction of the ε-NH2-Lys-135 with the sulfonamide, although alternative binding modes were seen here as well (Figure S3). Interestingly, Arg23 and Tyr136 form steep walls at the entry of the C-9 cavity (Figure S3b). The sulfonamide is deposited at a low level directly in front of the cleft with ample options to further grow the ligand into the cleft (Figures 4 and S3). To the best of our knowledge, this is the first report of 9-Nalkylsulfonamide-substituted sialic acids. The ability to synthesize these derivatives paved the way to search for affinity enhancing substitutions within the vast field of alkylsulfonylamide derivatives. A recent report already showed that 9arylsulfonamide-substituted sialyltrisaccharides can form selective ligands for mouse Siglec-F and mCD33, which can be synthesized even directly from the unprotected sugars under Schotten−Baumann conditions.59 First, we synthesized extended alkylsulfonamides (Scheme 4). The affinity of ethylsulfonamide 33 increased by a factor of 2, whereas the affinity of propyl and butyl sulfonamides decreased (Table 6). The absolute IC50 values of 32 are in the range of 1−3 μM (Table S1 and Figure S4) and varied considerably less than the values of low affinity ligand 1. The

Table 4. The rIPs of Derivatives with Combined Residues at Positions 2 and 9

As shown above, α-Me Neu5Ac derivative 10, with a phenylacetyl substitution at position 9, has a 10-fold increased affinity over 1 (Table 1). Therefore, we synthesized the corresponding 2-α-biphenyltriazol analog 30 (Scheme 3). Surprisingly, the affinity dropped to a factor of 0.60. The reason for this is not yet clear and needs further investigation. We needed additional strategies on how to further improve the affinity of 17. We noticed that reported cocrystal structures show an interaction of ε-NH2-Lys-135 and the 9-hydroxy group of the ligand.34,50 Therefore, we hypothesized that a negatively charged substituent attached to the 9 amino group of 21 could form an ionic interaction with ε-NH2-Lys-135, despite of the solvent exposed and flexible side chain (Figure 2). To test this hypothesis, we synthesized the N-sulfate 31 by reacting amine 20 with a pyridine-SO3 complex under aqueous conditions E



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Scheme 4. Synthesis of 9-N-Sulfonylated 2-α-Biphenyltriazol Ligandsa

a

Reagents and conditions: (a) (i) 20, H2O, NaOH, pH 9, pyridine SO3, 17 h; (ii) NaOH, 84%. (b) 20, DCM, triethylamine, DMF, alkylsulfonyl chloride. (c) EtOH, H2O, NaOH, 63−78% (b plus c). (d) 28, DMF, H2O, arylsulfonyl chloride, 70−85% (d plus c). (e) 20, DCM, triethylamine, DMA, triflate anhydride, 41% (e plus c). (f) (i) 20, DCM, triethylamine, sulfamoyl chloride; (ii) NaOH, 37%. (g) (i) DCM, TEA, Cbz-sulfamoyl chloride; (ii) 28, CH3CN, 59% (i plus c). (h) MeOH, Pd/C, H2, 98%.

Table 5. The rIPs of the N-Sulfate Derivative 31

Table 6. The rIPs of Other Sulfonamide Derivatives

values represent the inhibition of soluble Siglec-7-Fc protein binding to RBC-bound ligands and are not absolute binding constants. We started to explore the biological functions of Siglec-7 on NK cells using 32, although we did not know if the compound was able to block binding of NK cell membraneexpressed Siglec-7 to target cell ligands at the same IC50 values. Second, and in parallel to performing biological experiments, we synthesized additional compounds with aromatic sulfonylamides in order to address the C9-cavity and to benefit from additional interactions with Tyr-136 or Arg-23 (Figures 1 and 4). Arylsulfonyl chlorides were reacted with 20 under Schotten−Baumann conditions. Subsequent saponifications produced 36, 37, and 38 (Scheme 4). Benzylsulfonyl chloride was reacted with the hydrochloride 28 to yield 39 after saponification (Scheme 4). Additionally, Cbz-sulfamoyl chlor-

ide was used to form a Burgess-type reagent60,61 which reacted well with amine 28. Subsequent saponification produced 40 (Scheme 4). The affinity assay showed decreased binding (Table 7), despite promising results in docking experiments with the aromatic residue stacked within the C9-cavity in extension of the glycerol chain (not shown). A detailed analysis of the C9-cavity showed two bound water molecules in all six crystal structures (Figures S5 and S6). At least one of these can F



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that most of the affinity gain might be attributable to one of the sulfonyl oxygens (Table 8). Table 8. The rIPs of Other Sulfonamide Derivatives

Figure 4. Exemplary docking result of PDB entry 2HRL* with 32.

Table 7. The rIPs of Aromatic Sulfonamide Derivatives

To explore the effects of the sulfonyloxygens, subsequently, we performed docking experiments with the virtual compound 9-N-sulfate α-O-Me Neu5Ac 44 (Figure 5a) and made an overlay with the corresponding oxamido ligand 2 within the crystal structure 2G5R (Figure 5b). An almost exact spatial overlay of the second carbonyl oxygen of oxamide 2 and one sulfonyl oxygen of 44 is evident. This oxygen forms a hydrogen bond to the backbone nitrogen of Lys-135 (Figure 5b).

be seen as an integral part of the protein due to many possible hydrogen bonds with the protein backbone.62 The second one seems to be more suitable for replacement by ligands, but addressing such a cavity can be very challenging, as observed for PDE10 inhibitors.62 The rational design of sulfonamide extensions that can replace at least one of the two water molecules requires a much more detailed knowledge of the exact binding mode of 31 or 33 and the protein conformation in complex with the former. We investigated other small sulfonamides triggered by the findings that differences in polarity and charge of the N-sulfate 31 and the methylsulfonamide 32 did not greatly influence the affinity and that larger residues did not result in enhanced affinity. First, hydrochloride 28 was reacted with triflic anhydride and subsequently saponified to yield the trifluoromethylsulfonamide 41 (Scheme 4). Second, sulfamoyl chloride was reacted under basic conditions63 with 28 and saponified to yield the negatively charged sulfamoylazadinylsulfonamide 43 (Scheme 4). Third, the benzyloxycarbonyl group of previously synthesized 40 was removed via hydrogenation to obtain sulfamide 42 (Scheme 4). Despite the structural diversity, the affinity did not change much, indicating

Figure 5. (a) Virtual ligand 9-N-sulfate 2-α-Me Neu5Ac 44. (b) Overlay of virtual, sulfated ligand 44 (licorice; docking result with protein part of PDB entry 2G5R) and cocrystal bound oxamide ligand 2 (gray lines; PDB entry 2G5R). G



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Table 9. The rIPs and Kd Value Extrapolation of 32 and 33a

a

The asterisk denotes the extrapolated value; calculated by dividing the Kd value of 3′SLL by the ELISA rIP value of 32 or 33, respectively; n.a.: not applicable; n.d.: not determined.

other high-affinity ligands. Interestingly, Siglec-7 was described as an antiproliferative, apoptosis-inducing receptor on myeloid cells and myeloid cancers upon treatment with antibodies blocking the ligand binding site.67,68 In order to exclude effects mediated by mechanisms apart from the blocked Siglec-7 receptor−ligand interaction, we analyzed whether the inhibitor caused toxicity toward the NK and target cells used. Therefore, IL-2 prestimulated NK cells were incubated for 24 h in the presence of the Siglec-7 inhibitor and subsequently analyzed by a live−dead stain for the abundance of dying cells. In those experiments, the inhibitors did not cause any change in the viability of the analyzed NK cells. The NK cells remained fully viable even at concentrations up to 10 mM (data not shown).69 Notably, tumor target cells in the presence of the inhibitor alone also did not suffer from increased lysis compared to medium controls (data not shown). Taken together, these findings demonstrate that 32 has no toxic effects on cells in vitro. In addition to Siglec-7, human NK cells also express the closely related receptor Siglec-9, albeit at lower expression levels (Figure 6A).13,70−73 We therefore wanted to check for a possible cross-reaction of our Siglec-7 inhibitor with Siglec-9. The two proteins have 76% similarities in their ligand binding domain and display different ligand preferences only based on a six amino acid sequence.36,74 These amino acids form in part the Tyr-64 cavity, and one might therefore predict selectivity of our ligands toward Siglec-7. However, we still wanted to experimentally explore the affinity of 32 to Siglec-9, because such an interaction might cause functional effects even in absence of any impact on Siglec-7.13 Human erythrocytes used for the affinity testing do not express reasonable amounts of Siglec-9 ligands (Figure 6B). To test for the inhibition of Siglec-

Together with the hydrogen bonds of 9-NH to CO-Asn-133, 7OH to NH-Asn-133, and 5-NH CO-Lys-131, four hydrogen bonds between ligand and protein backbone are formed. The fourth hydrogen bond can be very beneficial for affinity due to cooperative effects.63,64 Favorable geometries of the sulfonamide within the hydrogen-bond network could be the cause of the increased affinity. In addition to the standard assay, we confirmed the results for the best ligands in a secondary, ELISA-based assay.31 In brief, biotinylated 6′sialyllactosamine-polyacrylamide is immobilized onto a Streptavidin-coated plate. Anti-hIgG-alkaline phosphatase precomplexed Siglec-7-Fc and serial dilutions of the ligands were added and incubated for 2.5h. The plate was washed and incubated with 4-nitrophenyl phosphate, and absorbance was quantified at 405 nm. The assay could not determine the IC50 values for 1, which were found to be >50 mM, but we could use known Siglec-7 ligands 3′sialyllactose and 6′sialyllactose as references.66 The advantages of these substances are the known absolute binding constants (Kd values).66 Indeed, we could confirm the high affinity of 32 and 33 in this assay. In addition, we could show that 32 and 33 display one digit micromolar Kd (Table 9). Overall, the best ligand identified so far is 33, displaying more than a 5000-fold enhanced affinity over the starting scaffold 1 and a Kd value in the low micromolar range. Further investigations to verify the binding mode for high-affine sulfonamide ligands are underway. Biology. We set out to analyze the potential to affect NK cell function as soon as we had the first high-affine Siglec-7 ligands at hand. We chose 32 because we had sufficient amounts of compound available and the derivative does not show any structural disadvantages for in vitro assays over the H



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Mel1106 as a target cell. This tumor cell line expresses Siglec-7 and Siglec-9 ligands (Figure 7A) and can be lysed by human

Figure 6. Inhibition of Siglec-7 and Siglec-9 by 32. (A) IL-2-activated NK cells were stained with anti-Siglec-7, anti-Siglec-9, or isotype control antibodies as indicated. (B) Cells were stained using Siglec-7Ig or Siglec-9-Ig fusion proteins to detect Siglec ligand expression on RBCs. (C, D) Inhibition of Siglec binding to K562 by 32. K562 cells were stained using Siglec-7-Ig or Siglec-9-Ig in the presence of the indicated concentrations of the inhibitor 32. Inhibition of Siglec-Ig fusion protein binding was calculated as described for the RBC affinity assay. All data shown are representative of at least three independent experiments.

Figure 7. Impact of Siglec-7 inhibition on NK cell function. (A) Mell1106 cells were stained with Siglec-7-Ig fusion protein to detect Siglec-7 ligand expression. The gray area shows secondary antibody staining as a control. (B, C) Effect of 32 on killing of Mel1106 by NK cells. IL-2-activated NK cells were incubated in the presence of inhibitor 32 or 1 as a control (both at 0.3 mM) with Mel1106 in a 4 h 51 Cr release assay. E:T means ratio of effector cells to target cells. (B) Example of a nonresponding NK cells. (C) Example of a responding NK cells. Data are representative mean values of at least three independent experiments ± SD for each NK isolation.

9, we therefore used the cell line K562 which expresses ligands for both receptors, Siglec-7 and Siglec-9 (Figure 6C). Fc-fusion proteins of both receptors were pre-incubated with the Siglec-7 inhibitor and then co-incubated with K562 cells in a modified affinity assay protocol. These cells were then analyzed by flow cytometry for residual binding of the fusion proteins to the expressed Siglec ligands. In those assays, the Siglec-7 inhibitor was shown to be about 100-fold more effective for Siglec-7 than for Siglec-9 (Figure 6C,D). Notably, the IC50 values were in the same range as observed for the RBCs. This demonstrates the specificity of our inhibitor. To test for the functional activity of our inhibitor, we performed chromium release assays with human NK cells to analyze their cytotoxic activity. Human NK cells were isolated and prestimulated with IL-2 prior to incubation with the Siglec7 inhibitor or controls. We used the melanoma cell line

NK cells in a standard 4 h chromium release assay (Figure 7B). The lysis could be increased after pre-incubation of the NK cells with 32, suggesting that the inhibitor interferes with the inhibitory effect of Siglec-7. Interestingly, this effect was donor dependent and only observable in about 50 percent of the samples (Figure 7B,C). This could be due to donor-specific expression of distinctly sized Siglec-7 isoforms,1,2 donor specific expression patterns of other inhibitory receptors overriding the Siglec-7 effect, or donor specific differences of NK cell surface expressed cis-ligands. Importantly, Siglec-7 is cis masked by endogenous ligands on NK cells,5,42 and sialidase treatment of NK cells unmasks Siglec-7, leading to a more pronounced inhibitory effect of I



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Siglec-7.28,30,42 In consequence, the effect of blocking Siglec-7 by antibodies after sialidase treatment was found to be much higher.30,42,75 We did not perform sialidase treatments, because global removal of sialic acids from the cell surface can have a broad demasking effect on other sialic acid recognizing NK cell receptors, such as Siglec-9, NKp46, NKG2D, NKG2A, and NKG2C, overriding the Siglec-7 ligand specific effect.71,73,76−78 Nevertheless, we hypothesize that under certain physiological conditions, including malignancies, NK cell bound Siglec-7 is demasked by specific sialidases to potentiate its inhibitory functions. The data shows for the first time that small soluble Siglec-7 ligands can be used to increase NK cell-mediated lysis of cancer cells, although reported values of increased lysis upon treatment of NK cells with anti-Siglec-7 antibodies were found to be higher than for our compound 32.13,30 This can be explained by the low micromolar range Kd value of 32, whereas antibodies normally have Kd values in the low nanomolar range and therefore are up to a 1000-fold more potent. In addition, it has to be considered that compound 32 has to prevent clustering of Siglec-7 at sites of cell contacts, which can be a more complex task than to outcompete soluble Siglec-7-Fc binding to RBC sialic acids. Second-generation compounds with low nanomolar Kd values might have much stronger effects on NK cellmediated lysis.

Biotinylated goat-α-human IgG-Fc antibody and the streptavidinphycoerythrin conjugate were obtained from Jackson Immuno Research, West Grove, PA. IgG control PE (MOPC21) was obtained from BioLegend, San Diego, CA. Murine α-Siglec-7-PE antibody (F023-420) was obtained from Pharmingen, BD, Franklin Lakes, NJ. Murine α-Siglec-9-PE antibody (191240) was obtained from R&D Systems, Minneapolis, MN. Cell Culture. All cells were grown at 37 °C and 5% CO2 in a humidified incubator under sterile conditions. Cell lines were split regularly either by medium exchange (K562) or trypsinization (Mel1106) every 2−3 days, and cell culture flasks were replaced regularly. RBC Isolation. RBCs were obtained from whole blood or buffy coats: 2−3 mL blood were washed with 10 mL PBS and centrifuged at 3000g for 10 min at room temperature. Two mL of the pelleted RBCs were harvested, washed twice with 13 mL of RBC wash buffer, and centrifuged at 2000g for 10 min at room temperature. A 0.1% (v/v) RBC solution in RBC wash buffer was prepared with the final pellet and stored at 4 °C for further experiments. NK Cell Isolation and Stimulation. Human polyclonal NK cells were isolated from whole blood or buffy coats. PBMCs were separated from the remaining cells by a density gradient centrifugation of the blood over lymphocyte separation medium. PBMCs were then harvested from the gradients and washed. NK cells were isolated from the PBMCs by the use of a NK cell negative isolation kit (Invitrogen, Carlsbad, CA). NK cell yield (NKp46+, CD3−, CD56+ cells) ranged from 90−99% of the purified cells. Purified cells were maintained in primary NK cell medium (IMDM, 10% (v/v) human serum, 1% (v/v) NEAA, 1% (v/v) sodium pyruvate) at a concentration of 2 × 106 cells/mL. Prior to use in experiments, NK cells were stimulated with 100 IU/mL hIL-2 for 24 h. FACS Analysis. 1 × 105 cells were resuspended in FACS buffer with the corresponding labeled antibody and were incubated for 20 min at 4 °C in the dark. Cells were then washed with FACS buffer, pelleted by centrifugation at 480g for 5 min at 4 °C, and resuspended FACS buffer containing 2% formaldehyde for fixation. Cells were then analyzed using a BD FACScalibur device, and results were evaluated and presented with the FlowJo software from Treestar. All histograms were presented as % of max. FACS-Based Inhibition Assay. The assay was adapted from a previous Siglec-7 study and modified to fit our requirements.34,39 Dilution series of the Siglec inhibitors were prepared in PBS. In a total of 50 μL PBS, 0.2 μg/mL Siglec-7-Fc and variable concentrations of the corresponding inhibitor were incubated for 10 min at RT. Meanwhile, 50 μL of the previously prepared 0.1% RBC solution was pelleted by centrifugation at 2000g for 5 min at 4 °C. RBCs were resuspended in the ligand/Siglec-7-Fc mixture and incubated for additional 20 min on ice to allow binding of free Siglec-7-Fc to RBC. The RBCs were washed with 150 μL FACS buffer and centrifuged at 2000g for 5 min at 4 °C. For detection of bound Siglec-Fc, the RBCs were resuspended in 50 μL FACS buffer with 10 μg/mL biotin conjugated goat-α-human-Fc antibody and incubated for 20 min on ice. The RBCs were then washed and pelleted, resuspended in 50 μL FACS buffer with 1:200 Streptavidin-PE, and incubated for 20 min on ice in the dark. Finally, the RBCs were washed and pelleted, resuspended FACS buffer with 2% formaldehyde, and analyzed on a BD FACScalibur device. In modified experiments, K562 (Figure 6C,D) or Mel1106 (Figure 7A), instead of RBCs, were mixed with the ligand/Siglec-Fc mixture in order to detect Siglec ligands on these cells. Subsequent steps in these assays were identical to the original inhibition assay. Data were evaluated with the FlowJo software. Percent inhibition of Siglec binding to RBCs by the novel compounds was calculated from the mean fluorescence intensity (MFI) values, whereby the MFI of unstained RBCs was set as 100% inhibition, and staining without inhibitor was set as 0% inhibition.

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CONCLUSIONS Cancer cells camouflage themselves with Siglec-7 ligands to evade the immune system. In particular NK cell-mediated lysis is blocked upon binding of NK cell expressed Siglec-7 to target cell surface-bound ligands, most probably due to recruitment of Siglec-7 to sites of cell−cell contacts.30 These facts induced our hypothesis that soluble Siglec-7 ligands can interfere with the Siglec-7-mediated inhibition of cancer cell lysis via NK cells, thereby providing a boost to the natural cancer immune response. The ligand binding site of Siglec-7 is highly flexible, and therefore, structure-guided design is challenging. We started from the minimal natural Siglec-7 ligand α-Me Neu5Ac and undertook various cycles of synthesis, computational predictions, and affinity assays to find better ligands. We obtained compounds with low miromolar Kd values and up to 5000-fold increased affinity over the starting scaffold and minimal natural Siglec-7 ligand α-Me Neu5Ac 1 based on the exploitation of unexpected results. The novel compounds are the first monovalent, low molecular weight Siglec-7 ligands with sufficient affinity to explore functional effects of inhibiting Siglec-7. We tested the effect of ligand 32 to promote cancer cell lysis by freshly isolated NK cells. The observed effects clearly support the hypothesis that soluble Siglec-7 ligands enhance NK cellmediated lysis of cancer cells, paving the way toward a novel immuno-oncology concept to enhance natural anticancer immune responses with low molecular weight compounds. Future studies of the exact binding modes will enable the design of next-generation Siglec-7 ligands, which hopefully can further verify their utility as broad anticancer agents in vivo.

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EXPERIMENTAL SECTION

Biology. Biological Materials. RBC wash buffer consisted of PBS with 0.1% (w/v) BSA and 2 mM EDTA. FACS buffer consisted of PBS with 2% (v/v) FBS. Human Siglec-7-Fc and Siglec-9-Fc fused to human IgG1 were obtained from R&D Systems, Minneapolis, MN.

%inhibition = 100 − ((MFI stained RBCs with inhibitor − MFI unstained RBCs/MFI)stained RBCs) × 100 J



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The IC50 values were determined by four parameter logistic fitting of the data with Origin software. ELISA-Based Inhibition Assay. ELISA plates (Maxisorb, Nunc) were coated with 1 mg/mL streptavidin in 50 mM NaHCO3 (pH 8.5) at 4 °C overnight. The next day, plates were washed 3 times with PBS, and 4 μg/mL 6′sialyl-N-acetyllactosamine-polyacrylamid-biotin (Lectinity) in PBS was added for 1 h at 37 °C. Afterward plates were blocked with 0.5% BSA in PBS for 3 h at 37 °C. Then plates were washed 3 times with PBS. The synthetic glycan ligands were diluted in PBS (10-fold dilutions from 1 mM to 1 × 10−5 mM). A complex of hSiglec7-Fc (1 mg/mL) with alkaline phosphatase-conjugated anti Fcantibody (anti-hIgG-AP, Jackson ImmunoResearch 1:750 dilution) was set up. Fifteen μL of the corresponding sialoside concentration and 15 μL of the hSiglec7-Fc complex were added to each well. The plates were incubated for 2.5 h at 37 °C. Then plates were washed 3 times with PBS 0.05% and Tween-20. The plates were developed with 1 mg/mL p-nitrophenyl phosphate in 1 M diethanolamine and 0.5 mM MgCl2 (pH 9.8). The fluorescence was determined at 405 nm in an ELISA reader, and IC50 values were calculated with the program SoftMaxPro. Chromium Release Assay. In chromium release assays, Mel1106 cells were used as target cells, whereas IL-2-activated human NK cells were used as effector cells. Target cells were grown to mid log phase, then 5 × 105 cells were harvested, resuspended in assay medium (IMDM medium containing 10% FBS and 1% Penicillin/Streptomycin), and labeled for 1 h at 37 °C with 100 μCi 51Cr (3 MBq). Target cells were washed twice and resuspended in fresh medium with a concentration of 5 × 104 target cells/mL. Siglec inhibitor dilutions were prepared in 96-well U-bottom plates, and effector cells were incubated with the inhibitors for 15 min prior to target cell addition. The 5 × 103 target cells were then added to all wells, and the plates were incubated for 4 h at 37 °C with 5% CO2. For maximum 51Cr release, labeled target cells were incubated with 1% Triton X-100, and for spontaneous 51Cr release, labeled target cells were incubated with medium alone. After the 4 h incubation, supernatants were harvested, and chromium release was measured in a γ-counter. All combinations were tested in triplicates. The percentage of specific release in the assay was calculated as follows: %specific release =

nitroprusside solution and dried on air. Details are available in Anfärbereagenzien für Dünnschicht and Papierchromatographie, Merck, 1970. Hydrogenations were carried out in a hydrogen atmosphere 50 mbar above atmospheric pressure. Optical rotations were measured on a Perkin Elmer Polarimeter 241. Normal phase flash chromatography was carried out using silica gel 60 (Merck, Geduran). For reversed phase flash chromatography, a glass column of 10 mm diameter was filled with 5 mL RP silica (YMC CO LTD., YMC ODSAQ). Due to the hydrophilicity of the gel, it was possible to run columns like normal phase silica columns, using low pressure generated with a hand driven pressure ball. A H2O to EtOH gradient was used for standard purifications. Columns were washed with EtOH and reused up to 30 times. All products containing an unprotected carboxylic acid were adjusted to pH 8−9 with NaHCO3 or Na2CO3 solution before loading onto the column to assure complete elution as sodium salt. Otherwise the column functioned, although in very small scale, as an ion exchanger, resulting in elution of small amounts (

Design, Synthesis, and Biological Evaluation of Small, High-Affinity

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